Epstein-Barr Virus in Somatic Cell Hybrids between Mouse Cells and Human Nasopharyngeal Carcinoma Cells ZENON STEPLEWSK1,I HILARY KOPROWSKI,' MARIA A N D E R S O N - A N V R E T AND GEORGE KLEIN ' The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Phcladelphra, Pennsylvania 19104,U S A . , and 'Department of Tumor Biology, Karolinska Institutet, S-10401 Stockholm 60,Sweden
ABSTRACT Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in t h e hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome. Studies of somatic cell hybrids between simian virus 40 (SV40)3-transformed human cells and mouse cells either of primary origin (Croce and Koprowski, '74a) or from established cell lines (Croce et al., '73; Croce and Koprowski, '74b) t h a t segregate human chromosomes, permitted t h e assignment of SV40 integration sites (Croce, '77; Khoury and Croce, '75; Croce et al., '75) to human chromosomes 7 and 17 in different SV40-transformed human cells. Results of these studies prompted us to extend our investigations to human tumors, particularly those that may be caused by viruses. Somatic cell hybrids derived from the fusion of Burkitt lymphoma cells with either mouse or human cells have been studied previously (Glaser and O'Neill, '72; Glaser and Rapp, '72; Glaser and Nonoyama, '73; Klein et al., '74). The mouse-human hybrids t h a t segregated human chromosomes did not express such Epstein-Barr virus (EBV) antigens as early antigen, virus capsid antigen, or membrane antigen; but they did express Epstein-Barr nuclear antigen (EBNA), the only antigen regularly found in proliferating, EBV-carrying cells. Loss of human chromosomes in human-mouse hybrids was accompanied by a decrease in the number of cells showing EBNA-positive nuclei; when hybrid cells contained only two to five human chromosomes, they did not show nuclear staining for EBNA (Klein et al., '74). J. CELL. PHYSIOL. (1978) 97: 1-8.
Cells of nasopharyngeal carcinoma (NPC) show EBNA-positive nuclei and the presence of multiple copies of EBV-DNA per cell (Klein, '75; zur Hausen et al., '70; Nonoyama et al., '73; Klein et al., '76; Kaschka-Dierich et al., '76). In addition, tumor-bearing patients have a high titer of antibodies against EBV-specific antigens (Klein, '75; Henle et al., '70, '73; De Schryver et al., '69; de The et al., '73, '75). The purpose of our study was to produce somatic cell hybrids between the cells derived from NPC biopsies and mouse cells in order to determine whether the expression of EBNA in the hybrid cells related to the retention of a specific human chromosome(s) derived from patients bearing NPC. MATERIALS A N D METHODS
Tumors Biopsies of NPC were obtained from Nairobi and shipped on wet ice to Philadelphia immersed in tissue culture medium. Upon arrival a t The Wistar Institute, tumor fragments were minced into very small pieces, which were immersed in 30-50 ml of L15 medium containing 20%fetal calf serum and stirred for 20-30 minutes a t room temperature to separate free tumor cells. The cells were then colReceived Feb. 10. '78. Accepted Mar. 7, '78. Abbreviations used in this paper are: SV40, simian virus 40; EBV, Epstein-Barr virus; EBNA, Epstein-Barr nuclear antigen; NPC. nasopharyngeal carcinoma; HPRT, hypoxanthine phosphoribosyltransferase; HAT, hypoxanthine-aminopterin-thymidine rnediurn; and cRNA, complementary RNA.
1
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STEPLEWSKI, KOPROWSKI, ANDERSSON-ANVRET AND KLEIN
lected by centrifugation, resuspended in serum-free medium and used for fusion experiments.
NPC transplanted in nude mice Two NPC tumors (M and G) transplantable in nude mice were kindly supplied by Doctor B. C. Giovanella (Klein et al., '76). These tumors are maintained in nude mice by subcutaneously transplanting small minces of tumor from one mouse to five other nude mice. Mouse cells LM-TK-C1 1D (Dubbs and Kit, '64) and IT22 (Santachiara e t al., '70) cells, both deficient in thymidine kinase (TK; EC 2.7.1.211, and THO-2 cells (BALB/c), deficient in hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) and resistant to ouabain (Jha and Ozer, '76) were used for hybridization purposes. Cell fusion Human and mouse cell fusion was mediated by inactivated Sendai virus in suspension as described previously (Steplewski and Koprowski, '70). Selection of hybrids After fusion, cells were maintained in hypoxanthine-aminopterin-thymidine(HAT) medium (Littlefield, '64) which inhibited growth of the parental mouse cells. The parental NPC cells did not grow in culture. On occasion, ouaM (Croce, '77) bain a t a concentration of was added to the selective medium in order to destroy human fibroblasts, which were present in NPC tumor fragments. After 4-6 weeks, the colonies of hybrid cells were removed by trypsinization (mass culture) and cloned in microtiter plates.
Cloning and subcloning Cloning and subcloning were performed by seeding single cells in microtest plates (6-mm, Linbro). Cell suspensions containing five to ten single cells per 1ml were used to seed 0.2 ml aliquots/well. The next day, wells containing single cells were marked and the cells were collected as clones or subclones after sufficient number of cells formed a colony. EBNA test Tests for EBNA were carried out on smear preparations, fixed in methanol-acetone, ac-
cording to Reedman and Klein ('73). At least two EBNA antibody-positive and two negative sera were included in each test. Each hybrid culture or derived clone was tested on a t least three and sometimes ten or more independent occasions.
Nucleic acid hybridization tests The details of t h e procedures used for isolation of DNA and filter hybridization of cellular DNA to precalibrated EBV complement a r y RNA (cRNA) have been described elsewhere (Lindahl et al., '76). Briefly, 10-pg DNA aliquots were immobilized onto nitrocellulose membrane filters and incubated with 1 ng of EBV 32PcRNA in 0.3 ml of 0.9 M NaCl/O.O9 M trisodium citrate/50%formamide for four days a t 46OC. The filters were then washed at 60°C and treated with RNase before 32Pradioactivity was determined. The DNA content on each filter after hybridization was determined by the diphenylamine reaction, and the information was used to adjust the data to 10.0 p g of DNA per filter. The molecular weight for EBV-DNA was assumed to be 1 x lo8 and t h a t for cellular DNA to be 4 x 1012. Karyologic analysis Chromosomes of parental, mouse, and hybrid cells were identified by the Giemsa banding method by a modified procedure described by Seabright ('71). Isozyme analysis Hybrid clones were assayed for the expression of at least one isozyme assigned to each human chromosome except for chromosomes 22 and Y (McKusick and Ruddle, '77) (The Third International Workshop on Human Chromosome Mapping, Baltimore, October, 1975). Back selection Two EBNA-positive clones, G8/I-10 and D12/II, were backselected in medium containing 100 pg/mi of BrdU as described previously (Croce, '77). RESULTS
Production of hybrid cultures Since cells derived from NPC tumors cannot be propagated in tissue culture, we fused NPC cells derived directly from the tumor with the three drug-resistant mouse cell lines. Thir-
3
HUMAN NPC AND EBV IN SOMATIC CELL HYBRIDS TABLE 1
Hybrids’ established after fusion of NPC tumor cells with mouse cells Orion of tumor cells
Hybrids with mouse cells C1 1D
Patients NN (935595) WK (159866) KM-l(165836) KM-2 (163859) AM (16628) EJ (94057) JN (170788)
Nude mice M G
IT22
THO-2
Number of human chromosomes present
NN-T-2 NN-T-3 WK-D-1 WK-D-2
-
8-15
212
43
212
5-8 5-8 6-10
111 111 113
5-8 5-8
111 111
4 12-15
011 111
-
KM-1-T-9 KM-2-T-10 AM-T-11 AM-T-12 AM-T-13
-
-
Ratio of EBNA-positive hybrid cultures
EJ-H-1 JN-H-I
‘ M-T-8 G-T-7
’ As defined by growth in HAT medium and presence of human chromosomes (see text). As determined by karyotypic analysis or Karyotypic and isozyme analysis. NPC tumors proven to he EBNA-positive and to contain EB-viral DNA (14) were grown in nude mice.
teen independent hybrid lines (table 1) were developed; 11from t h e fusion of NPC cells obtained from seven patients with NPC tumors and two from the two NPC tumors maintained by serial passages in nude mice (Klein et al., ’76). These hybrids grew in HAT medium, and karyotypic analysis of the cells showed the presence of a number of human chromosomes and the entire complement of chromosomes of mouse parental cells. Presence of human chromosomes in cells of cultures derived from fusion of WK NPC cells with C1 ID cells (table 1) was determined by karyologic and isozyme analysis (see below). Of t h e 13 hybrid cultures, ten contained cells with EBNA-positive nuclei (table 1).
The percentage of EBNA-positive cells in the WK-D-1 mass culture a t the level of consecutive tissue culture passages decreased from 80-90% to 20-30% after six to eight months. In contrast, mass hybrid culture NNT-3 retained 80% EBNA-positive cells after more than one year’s growth in vitro. Clones and subclones of hybrid culture WK-D-1 showed less of a decrease in the percentage of EBNA-positive cells than did the mass culture. For instance, the percentage of EBNApositive cells in three WK-D-1 clones decreased from 70% to 50%in the course of eight months’ growth in tissue culture. Subclones of EBNA-positive clones of hybrid WK-D-’1 varied somewhat in their ability to retain EBNA-positive cells. In some subclones, a high percentage (90%)of EBNA-positive cells was maintained for more than a 6-month observation period, whereas in other subclones the percentage of E-positive cells decreased to 10-20%during the same time period.
Maintenance of EBNA-positive cells in mass hybrid cultures, clones and subclones Of 19 clones derived from hybrid culture WK x C1 D (WK-D-1),14showed EBNA-positive cells. Of 40 subclones obtained from three EBNA-positive WK-D-1 clones, 34 contained Attempts to associate presence of a specific EBNA-positive cells. All of the 21 clones dehuman chromosome with the rived from hybrid culture NN x IT-22 (NN-Texpression of EBNA 3) contained EBNA-positive cells. Six of 22 Karyotypic analysis of hybrids with mouse clones derived from hybrid culture KM-2 x IT-22 (KM-2-T-10)were analyzed for presence IT-22 and THO-2 cells showed, in addition to of EBNA-positive cells, and only one clone an entire complement of mouse chromosomes, showed presence of such cells. Of 28 clones de- five to eight human chromosomes, of which rived from hybrid culture G X IT-22 (G-T-7), chromosomes 7, 16, 17, and 21 in hybrids KM1-T-9and KM-2-T-10 and chromosomes 19 and 1 4 contained EBNA-positive cells.
LW-A
AK-
1 GOT-1
GUS GRS
PGM-3
SOD-2
HM-B
I M -1 GALT 2GM-2
PGM- 1
phosphog luconiuta s e- 1 (chromosome 1) isocitra te dehydroqenase-1 (chromosome 2 ) galactose-1-phosphate uridiltransferase (chromosome 3 ) phosphoglucornutase-2 (chromosome 4 ) hexosaainidase B (chromosome 5 ) superoxide dismutase-2 (chromosome 6) phosphoqlucon~utase-3 (chromosome 6) beta-glucuronidase (chromosome 7) glutathione reductase (chromosome 8 ) adenylate kinase-1 (chromosome 9) glutamate oxaloacetic transaminase-1 (chromosome 10) lactate dehydrogenase A (chromosome 11)
Symbols used for the isozvme ( 2 8 ) :
C6 PD
NP MPI HEX-A APRT GX PEP-A PH I ADA SOD- 1
ESD
LDH -3 ENO- 2 PEP-B
lactate dehydroqenase B (chromosome 12) enolase-2 (chromosome 12) peptidase B (chromosome 12) esterase D (chromosome 13) nucleoside phosphorylase (chromosome 14) mannosephosphate isomerase (chromosome 15) hexosaminidase A (chromosome 15) adenine phosphoribosy-transferase (chromosome 16) galactokinase (chromosome 17) peptidase A (chromosome 18) phosphohexose isomerase (chromosome 19) adenosine deaminase (chromosome 23) superoxide dismutase-1 (chromosome 21) glucose-6-phosphate dehydrogenase (X chromosome!
Expression of human lsozymes in clones and subclones of WK D-1 hybrid
TABLE 2
HUMAN NPC AND EBV I N SOMATIC CELL HYBRIDS
X in hybrid JN were more frequently represented than other human chromosomes. The hybrid NN-"-3 and its clones showed the presence of 8 to 12 human chromosomes including chromosomes 5, 7, 10, 12, 17, 19, 20 and 21. Hybrid culture G-T-7 (derived from NPC propagated in nude mice) and its clones contained 80-100% cells staining positively for EBNA. Karyotypic analysis of metaphases and isozyme analysis of this hybrid indicated t h e presence of 12 human chromosomes: 1, 5, 6, 9, 14, 15, 16, 17, 18, 19, 20, and 21. A detailed analysis of WK-D-1 hybrid culture clones and subclones for the presence of EBNA, and t h e expression of human forms of isozymes, is shown in table 2. Results of karyological analysis confirm the isozyme results. Of five WK-D-1clones, two were EBNA-negative; results of isozyme analysis of these clones, C3/I and C8/I, are shown in table 2. Isozyme analysis of t h e three EBNA-positive clones, D12II1, G-4/11, and G8/I (table 2) indicated the presence of the following human chromosomes: 12 (ENO-2, PEP-B, and LDHB), 17 (GK), 20 (ADA), and 21 (SOD-1). Of eight subclones derived from clone G8/I, five contained EBNA-positive cells and three did not (fig. 1).Human chromosomes 12 and 21 were present in cells of three EBNA-negative and three EBNA-positive subclones. In addition, two EBNA-negative subclones contained human chromosome 20. All subclones contained human chromosome 17 and one of these subclones (G8/1-3), which contained 8090% EBNA-positive cells and only human chromosomes 17 and 21, was then subcloned. Three of the 14 resulting subclones contained EBNA-positive cells, but there was no correlation between the retention of human chromosomes 17 and 21 and expression of EBNA. In addition, one EBNA-positive subclone, G8/I-6
Human chromosomes 12
EBNA
21
t
-
4-
-
+
3
2
3
2
-
3
0
3
0
Fig. 1 Lack of association between the presence of
EBNA and expression of either human ENO-2 (chromosome 12) or SOD-1 (chromosome 21) in eight subclones derived from clone G8/I (see details in table 3).
5
(table 21, showed the presence of human chromosomes 12 and 17, but not 21. Finally, another EBNA-positive subclone, G8/I-10, showed the presence of only human chromosome 17 (table 2). Similar patterns were observed in one subclone, G4/II-12, derived from clone G4/II and two subclones, D12/11-1and D12/II-14,derived from clone D12III (table 2). All subclones contained EBNA-positive cells and retained, in addition to a n entire mouse complement, any combination of human chromosomes 12, 17, 20 and 21. Back selection in Brd U
To exclude the role of human chromosome 17 in t h e maintenance of EBNA, cells of clones G8/I-10 and D12III were transferred to medium containing BrdU (Croce, '77; Croce et al., '731, and the surviving colonies were propagated for three passages in BrdU medium before analysis. All four subclones established after back selection of clone GEYI-10 in BrdU became EBNA-negative and lost expression of human galactokinase. On the contrary, as shown in table 2, none of the nine BU subclones of clone D12/II expressed the human form of galactokinase, which indicates loss of human chromosome 17 and all these showed presence of EBNA-positivecells in proportions varying from 40-100%. All D12/II-derived subclones expressed human ENO-2 and three clones expressed the human form of PEP-B, which indicates the presence of the proximal fragment of the long arm of human chromosome 12 (McKusick and Ruddle, '77). In addition, four subclones expressed the human form of ADA which indicates the presence of human chromosome 20, and one subclone expressed SOD-1, which indicates presence of human chromosome 21. Clone D12III-BU-6, which contained 40.50% of EBNA-positive cells and expressed only human ENO-2 and PEP-B (table 21, was subcloned. As shown in the ideogram (fig. 3) 10 of the 13 subclones contained EBNA-positive cells and three did not. All 13 cultures expressed human ENO-2, indicating that in all cultures part of human chromosome 12 was present. EBVgenome equivalents in NPCand hybrid cells The results shown in table 3 indicate that, in general, the number of EBV-DNA equivalents per cell decreased in hybrid cells as
6
STEPLEWSKI, KOPROWSKI, ANDERSSON-ANVRET AND KLEIN TABLE 3
Equivalents of EBVgenomes per cell in human parental NPCcells and their hybrids Cells Parental NPC
"(935595)'
Hybrid mass cultures
Clones
EBV genome equivalents
Subclones
WK (159866)' WK-D-1
4
G4/II G8/I
11 10
G8II-3 G8II-4 GWI-9 GIII-I1 G8/I-14
KM-2 (163859)' AM (166628)' EJ (94057)'
G (nude)'
12 - 2 < 1 5 < 1 23 21 26 104 2 30 < 1 40 < 1 25 3 25 5 4 7
KM-1-T-9' KM-2-T-10' AM-T-11 ' EJ-H-1 '
JN (170788)'
+ + + + + + + +-
22 5 5
NN-T-3 '
KM-1 (165836)'
EBNA
JN-H-1 ' G-T-7 '
++
+
+
+
+
+
' See table 1 and text. *Seetable 2 and text. After more than one year in culture, these hybrids have recently become EBNA negative. EBV-DNA equivalents were assayed after hybrids became EBNA negative.
compared to their corresponding parental (patient's) tumor cells. WK-D-1 and G-T-7 hybrids are the exceptions since there was anincrease in t h e amount of EBV genome equivalents per cell in comparison to the parental NPC cells. All hybrid cells that contained EBV-DNA were found t o express EBNA, and the EBNA-negative subclones of hybrid WK-D-1 contained less than one EBV genome equivalent per cell. In two cases (KM2-T-10 and AM-T-111, hybrids that were EBNA-positive at the beginning have recently become EBNA-negative and contain less than one EBV genome equivalent per cell. Regardless of t h e presence or absence of EBV-DNA, all hybrids contained human chromosome(s). DISCUSSION
Although human chromosomes 12 and 21 (or parts of them) seemed to be preferentially retained in EBNA-positive hybrid cells, the results of subcloning these hybrids indicated that neither chromosome 12 nor 21 segregates with EBNA (fig. 1). Analysis of subclones of
Human chromosomes
17
21
EBNA
+
-
f
-
+
3
0
3
0
-
10
1
11
0
Fig. 2 Lack of correlation between presence of EBNA and expression of human SOD-1 (chromosome 21) and GK (chromosome 17) in 14 subclones derived from G8/I-3, an EBNA-positive subclone of WK-D-1 hybrid cultures between NPC and C1 1D cells (see text).
hybrid subclone G8/I-3 (fig. 2), which showed the presence of human chromosomes 21 and 17 as the only human chromosomes, confirmed the fact t h a t neither chromosome 21 nor 17 segregates with the expression of EBNA. Furthermore, in spite of the results obtained with back selection of clone G8/I-10, the possibility that human chromosome 17 maintains EBNA
HUMAN NPC AND EBV IN SOMATIC CELL HYBRIDS
7
Human chromosome 12
EBNA
-k
10
0
-
3
0
Fig. 3 Lack of correlation between presence of EBNA and expression of human ENO-2 in 13 subclones derived from t h e EBNA-positive DlB/II-BU-B subelone of the WK-D-1 hybrid culture between NPC and C1 1D (see text).
has been eliminated by examination of the subclones of hybrid DlBIII, which were backselected in BrdU and which expressed EBNA in the absence of human chromosome 17. Since all these subclones expressed human ENO-2, i t was hoped t h a t analysis of subclones of BU-6 (fig. 3) would permit the discovery of a more positive association between chromosome 12 and the presence of EBNA; the results proved the contrary and, as shown in figure 3, this association was not established for 3 out of 13 clones examined. Whether a fragment of human chromosome 12 was present in the three EBNA-negative subclones of clone G8/I (fig. 1) but could not be detected by isozyme analysis is impossible to exclude. Thus, in the light of these results, it is impossible to correlate the presence of EBVDNA and the expression of EBNA with a specific human chromosome(s), and we have to examine alternate mechanisms of maintaining EBNA in NPC-mouse hybrid cells. We may postulate that production of EBNA is associated with the presence of a fragment of a human chromosome carrying EBV viral DNA sequences which cannot be detected either by karyotype analysis or isozyme assays. Within t h e context of presently available information, it is not possible to disprove this rather unlikely hypothesis. The second possibility is that EBV is present in NPC cells in the form of free plasmids and, hence, can be found in NPC mouse hybrids regardless of the presence of a given human chromosome. However, data obtained by Falk e t al. (manuscript in preparation) indicate that one hybrid clone, NN-T2-7 may contain only integrated EBV-DNA sequences. A third possibility is that the expression of EBNA is due to the integration of t h e viral DNA sequences in the mouse chromosome. I t is clear from data presented in table 3 t h a t there is a positive correlation between
the presence of EBV-DNA and the expression of EBNA in the hybrid cells. On the other hand, there are hybrids that contain human chromosome(s) but are negative for EBNA and do not contain EBV-DNA. In contrast to the results obtained in studies of SV40-transformed human cells (Khoury and Croce, '75; Croce et al., '75) assignment of the gene(s) responsible for the maintenance of the malignant phenotype may present a much more difficult task in NPC hybrids. There is no reason, however, why these studies should not be pursued, since the study of interspecies somatic cell hybrids with a variety of human tumors could ultimately result in the identification of a human chromosome responsible for malignancy. ACKNOWLEDGMENTS
The NPC biopsies were collected and kindly supplied to G. K. by Doctor Surjit Singh (Department of Head and Neck Surgery, Kenyatta National Hospital, Nairobi, Kenya). The expert technical assistance of Maria Obrocka, Hanna Pragert and Marie Prewett is gratefully acknowledged. This investigation was supported, in part, by USPHS Research Grants CA-10815 from the National Cancer Institute and RR-05540 from the Division of Research Resources and by Grant 5 R 0 1 CA-14054-05 from t h e National Cancer Institute and a grant from the Swedish Cancer Society. LITERATURE CITED Croce, C. M. 1977 Assignment of the simian virus 40 integration site to chromosome 17 in t h e SV40-transformed human cell line GM54VA. Proc. Natl. Acad. Sci. (U.S.A.1, 74: 315-318. Croce, C. M., D. Aden and H. Koprowski 1975 Somatic cell hybrids between mouse peritoneal macrophages and simian virus 40 transformed human cells. 11. Presence of human chromosome 7 carrvine simian virus 40 eenome in cells of tumors induced b i h y i r i d cells. Proc. Natl. Acad. Sci. (USA.), 72: 1397-1400. Croce, C. M., A. J. Girardi and H. Koprowski 1973 Assign-
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STEPLEWSKI, KOPROWSKI, AN1DERSSON-ANVRET AND KLEIN
ment of the T antigen gene of simian virus 40 to human chromosome C-7. Proc. Natl. Acad. Sci. (U.S.A.), 70: 3617-3620. Croce, C. M., and H. Koprowski 1974a Somatic cell hybrids between mouse peritoneal macrophages and SV40-transformed human cells. I. Positive control of the transformed phenotype by th e human chromosome 7 carrying the SV40 genome. J. Exp. Med., 140: 1221-1229. 1974b Concordant segregation of the expression of SV40 T antigen and human chromosome 7 in mousehuman hybrid suhclones. J. Exp. Med., 139: 1350-1353. De Schryver, A,, S. Friberg, Jr., G. Klein, W. Henle, G. Henle, G. de The, P. Clifford and H. C. Ho 1969 Epstein-Barr virus-associated antibody patterns in carcinoma of the postnasal space. Clin. Exp. Immunol., 5: 443-459. de The, G., H. C. Ho, D. V. Ablashi, N. E. Day, A. J. L. Macario, M. C. Martin-Berthelon and R. Sohier 1975 Nasopharyngeal carcinoma. IX. Antibodies to EBNA and correlation with response t o other EBV antigens in Chinese patients. Int. J. Cancer, 16: 713-721. de The, G., R. Sohier, H. C. Ho and R. Freund 1973 Nasouharvneeal carcinoma. IV. Evolution of comdement . fixing antibodies during t he course of t he disease. Int. J. Cancer, 12: 368-377. Dubbs, D. R., and S. Kit 1964 Effect of halogenated pyrimidine and thymidine on growth of L-cells and a subline lacking thymidine kinase. Exp. Cell Res., 33: 19-28. Glaser, R., and F. J. ONeill 1972 Hybridization of Burkitt lymphoblastoid cells. Science, 176: 1245-1247. Glaser, R., and F. Rapp 1972 Rescue of Epstein-Barr virus from somatic cell hybrids of Burkitt lymphoblastoid cells. J. Virol., 10: 288-296. Glaser, R.; and M. Nonoyama 1973 Epstein-Barr virus: detection of genome in somatic cell hybrids of Burkitt lymphohlastoid cells. Science, 179: 492-493. Henle, W., G. Henle, H. C. Ho, P. Burtin, Y. Cachin, P. Clifford, A. De Schryver, G. de The, V. Diehl and G. Klein 1970 Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, other head and neck neoplasma and control groups. J. Natl. Cancer Inst., 44: 225-231. Henle, W., H. C. Ho, G. Henle and H. C. Kwan 1973 Antibodies to Epstein-Barr virus-related antigens in nasopharyngeal carcinomas. Comparison of active cases with long-term survivors. J. Natl. Cancer Inst., 51: 361-369. Jha , K. K., and H. L. Ozer 1976 Expression of transformation in cell hybrids. I. Isolation and application of densityinhibited Balb/3T3 cells deficient in hypoxanthine phosphorihoxyltransferase and resistant to ouabain. Somat. Cell Genet., 2: 215-223. .
I
Kaschka-Dierich, C., A. Adams, T. Lindahl, G. N. Bornkamm, G. Bjursell and G. Klein 1976 Intracellular forms of Epstein-Barr virus DNA in human tumor cells in vivo. Nature, 260: 302-306. Khoury, G., and C. M. Croce 1975 Quantitation of the viral DNA present in somatic cell hybrids between mouse and SV4O-transformed human cells. Cell, 6: 535-542. Klein, G. 1975 The Epstein-Barr virus and neoplasia. New Engl. J. Med., 293: 1353-1357. Klein, G., B. C. Giovanella, T. Lindahl, P. J. Fialkow, S. Singh and J. S. Stehlin 1976 Direct evidence for the presence of Epstein-Barr virus DNA and nuclear antigen in malignant epithelial cells from patients with poorly differentiated carcinoma of the nasopharynx. Proc. Natl. Acad. Sci. (U.S.A.), 74: 4737-4741. Klein, G., F. Wiener, L. Zech, H. zur Hausen and B. Reedman 1974 Segregation of the EBV-determined nuclear antigen (EBNA) in somatic cell hybrids derived from the fusion of a mouse fibroblast and a human Burkittlymphoma line. Int. J. Cancer, 14: 54-64. Lindahl, T., A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich and U. Jehn 1976 Covalently closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line. J. Mol. Biol., 102: 511-530. Littlefield, J. W. 1964 Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science, 145: 709-710. McKusick, V. A., and F. H. Ruddle 1977 The status of t h e gene map of the human chromosomes. Science, 196: 390-405. Nonoyama, M., C. H. Huang, J. S. Pagano, G. Klein and S. Singh 1973 DNA of Epstein-Barr virus detected in tissue of Burkitt's lymphoma and nasopharyngeal carcinoma. Proc. Natl. Acad. Sci. (U.S.A.). 70: 3265-3268. Reedman, B. M., and G. Klein 1973 Cellular localization of a n Epstein-Barr virus (EBVI-associated complement-fixing antigen in producer and nonproducer lymphoblastoid cell lines. Int. J. Cancer, 11: 499-520. Santachiara, A., M. Nabholz, V. Miggiano, A. J. Darlington and W. Bodmer 1970 Genetic analysis with man-mouse somatic cell hybrids. Nature, 227: 248-251. Seahright, M. 1971 A rapid banding technique for human chromosomes. Lancet, 2: 971-972. Steplewski, Z., and H. Koprowski 1970 In: Methods in Cancer Research. H. Bush, ed. Academic Press, New York, Vol. 5, pp. 155-191. zur Hausen, H., H. Schulte-Holthausen,G. Klein, W. Henle, G. Henle, D. Clifford and L. Santesson 1970 EBV-DNA in biopsies of Burkitt tumors and anaplastic carcinomas of. the nasopharynx. Nature, 228: 1056-1058.
ABSTRACT Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in t h e hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome. Studies of somatic cell hybrids between simian virus 40 (SV40)3-transformed human cells and mouse cells either of primary origin (Croce and Koprowski, '74a) or from established cell lines (Croce et al., '73; Croce and Koprowski, '74b) t h a t segregate human chromosomes, permitted t h e assignment of SV40 integration sites (Croce, '77; Khoury and Croce, '75; Croce et al., '75) to human chromosomes 7 and 17 in different SV40-transformed human cells. Results of these studies prompted us to extend our investigations to human tumors, particularly those that may be caused by viruses. Somatic cell hybrids derived from the fusion of Burkitt lymphoma cells with either mouse or human cells have been studied previously (Glaser and O'Neill, '72; Glaser and Rapp, '72; Glaser and Nonoyama, '73; Klein et al., '74). The mouse-human hybrids t h a t segregated human chromosomes did not express such Epstein-Barr virus (EBV) antigens as early antigen, virus capsid antigen, or membrane antigen; but they did express Epstein-Barr nuclear antigen (EBNA), the only antigen regularly found in proliferating, EBV-carrying cells. Loss of human chromosomes in human-mouse hybrids was accompanied by a decrease in the number of cells showing EBNA-positive nuclei; when hybrid cells contained only two to five human chromosomes, they did not show nuclear staining for EBNA (Klein et al., '74). J. CELL. PHYSIOL. (1978) 97: 1-8.
Cells of nasopharyngeal carcinoma (NPC) show EBNA-positive nuclei and the presence of multiple copies of EBV-DNA per cell (Klein, '75; zur Hausen et al., '70; Nonoyama et al., '73; Klein et al., '76; Kaschka-Dierich et al., '76). In addition, tumor-bearing patients have a high titer of antibodies against EBV-specific antigens (Klein, '75; Henle et al., '70, '73; De Schryver et al., '69; de The et al., '73, '75). The purpose of our study was to produce somatic cell hybrids between the cells derived from NPC biopsies and mouse cells in order to determine whether the expression of EBNA in the hybrid cells related to the retention of a specific human chromosome(s) derived from patients bearing NPC. MATERIALS A N D METHODS
Tumors Biopsies of NPC were obtained from Nairobi and shipped on wet ice to Philadelphia immersed in tissue culture medium. Upon arrival a t The Wistar Institute, tumor fragments were minced into very small pieces, which were immersed in 30-50 ml of L15 medium containing 20%fetal calf serum and stirred for 20-30 minutes a t room temperature to separate free tumor cells. The cells were then colReceived Feb. 10. '78. Accepted Mar. 7, '78. Abbreviations used in this paper are: SV40, simian virus 40; EBV, Epstein-Barr virus; EBNA, Epstein-Barr nuclear antigen; NPC. nasopharyngeal carcinoma; HPRT, hypoxanthine phosphoribosyltransferase; HAT, hypoxanthine-aminopterin-thymidine rnediurn; and cRNA, complementary RNA.
1
2
STEPLEWSKI, KOPROWSKI, ANDERSSON-ANVRET AND KLEIN
lected by centrifugation, resuspended in serum-free medium and used for fusion experiments.
NPC transplanted in nude mice Two NPC tumors (M and G) transplantable in nude mice were kindly supplied by Doctor B. C. Giovanella (Klein et al., '76). These tumors are maintained in nude mice by subcutaneously transplanting small minces of tumor from one mouse to five other nude mice. Mouse cells LM-TK-C1 1D (Dubbs and Kit, '64) and IT22 (Santachiara e t al., '70) cells, both deficient in thymidine kinase (TK; EC 2.7.1.211, and THO-2 cells (BALB/c), deficient in hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) and resistant to ouabain (Jha and Ozer, '76) were used for hybridization purposes. Cell fusion Human and mouse cell fusion was mediated by inactivated Sendai virus in suspension as described previously (Steplewski and Koprowski, '70). Selection of hybrids After fusion, cells were maintained in hypoxanthine-aminopterin-thymidine(HAT) medium (Littlefield, '64) which inhibited growth of the parental mouse cells. The parental NPC cells did not grow in culture. On occasion, ouaM (Croce, '77) bain a t a concentration of was added to the selective medium in order to destroy human fibroblasts, which were present in NPC tumor fragments. After 4-6 weeks, the colonies of hybrid cells were removed by trypsinization (mass culture) and cloned in microtiter plates.
Cloning and subcloning Cloning and subcloning were performed by seeding single cells in microtest plates (6-mm, Linbro). Cell suspensions containing five to ten single cells per 1ml were used to seed 0.2 ml aliquots/well. The next day, wells containing single cells were marked and the cells were collected as clones or subclones after sufficient number of cells formed a colony. EBNA test Tests for EBNA were carried out on smear preparations, fixed in methanol-acetone, ac-
cording to Reedman and Klein ('73). At least two EBNA antibody-positive and two negative sera were included in each test. Each hybrid culture or derived clone was tested on a t least three and sometimes ten or more independent occasions.
Nucleic acid hybridization tests The details of t h e procedures used for isolation of DNA and filter hybridization of cellular DNA to precalibrated EBV complement a r y RNA (cRNA) have been described elsewhere (Lindahl et al., '76). Briefly, 10-pg DNA aliquots were immobilized onto nitrocellulose membrane filters and incubated with 1 ng of EBV 32PcRNA in 0.3 ml of 0.9 M NaCl/O.O9 M trisodium citrate/50%formamide for four days a t 46OC. The filters were then washed at 60°C and treated with RNase before 32Pradioactivity was determined. The DNA content on each filter after hybridization was determined by the diphenylamine reaction, and the information was used to adjust the data to 10.0 p g of DNA per filter. The molecular weight for EBV-DNA was assumed to be 1 x lo8 and t h a t for cellular DNA to be 4 x 1012. Karyologic analysis Chromosomes of parental, mouse, and hybrid cells were identified by the Giemsa banding method by a modified procedure described by Seabright ('71). Isozyme analysis Hybrid clones were assayed for the expression of at least one isozyme assigned to each human chromosome except for chromosomes 22 and Y (McKusick and Ruddle, '77) (The Third International Workshop on Human Chromosome Mapping, Baltimore, October, 1975). Back selection Two EBNA-positive clones, G8/I-10 and D12/II, were backselected in medium containing 100 pg/mi of BrdU as described previously (Croce, '77). RESULTS
Production of hybrid cultures Since cells derived from NPC tumors cannot be propagated in tissue culture, we fused NPC cells derived directly from the tumor with the three drug-resistant mouse cell lines. Thir-
3
HUMAN NPC AND EBV IN SOMATIC CELL HYBRIDS TABLE 1
Hybrids’ established after fusion of NPC tumor cells with mouse cells Orion of tumor cells
Hybrids with mouse cells C1 1D
Patients NN (935595) WK (159866) KM-l(165836) KM-2 (163859) AM (16628) EJ (94057) JN (170788)
Nude mice M G
IT22
THO-2
Number of human chromosomes present
NN-T-2 NN-T-3 WK-D-1 WK-D-2
-
8-15
212
43
212
5-8 5-8 6-10
111 111 113
5-8 5-8
111 111
4 12-15
011 111
-
KM-1-T-9 KM-2-T-10 AM-T-11 AM-T-12 AM-T-13
-
-
Ratio of EBNA-positive hybrid cultures
EJ-H-1 JN-H-I
‘ M-T-8 G-T-7
’ As defined by growth in HAT medium and presence of human chromosomes (see text). As determined by karyotypic analysis or Karyotypic and isozyme analysis. NPC tumors proven to he EBNA-positive and to contain EB-viral DNA (14) were grown in nude mice.
teen independent hybrid lines (table 1) were developed; 11from t h e fusion of NPC cells obtained from seven patients with NPC tumors and two from the two NPC tumors maintained by serial passages in nude mice (Klein et al., ’76). These hybrids grew in HAT medium, and karyotypic analysis of the cells showed the presence of a number of human chromosomes and the entire complement of chromosomes of mouse parental cells. Presence of human chromosomes in cells of cultures derived from fusion of WK NPC cells with C1 ID cells (table 1) was determined by karyologic and isozyme analysis (see below). Of t h e 13 hybrid cultures, ten contained cells with EBNA-positive nuclei (table 1).
The percentage of EBNA-positive cells in the WK-D-1 mass culture a t the level of consecutive tissue culture passages decreased from 80-90% to 20-30% after six to eight months. In contrast, mass hybrid culture NNT-3 retained 80% EBNA-positive cells after more than one year’s growth in vitro. Clones and subclones of hybrid culture WK-D-1 showed less of a decrease in the percentage of EBNA-positive cells than did the mass culture. For instance, the percentage of EBNApositive cells in three WK-D-1 clones decreased from 70% to 50%in the course of eight months’ growth in tissue culture. Subclones of EBNA-positive clones of hybrid WK-D-’1 varied somewhat in their ability to retain EBNA-positive cells. In some subclones, a high percentage (90%)of EBNA-positive cells was maintained for more than a 6-month observation period, whereas in other subclones the percentage of E-positive cells decreased to 10-20%during the same time period.
Maintenance of EBNA-positive cells in mass hybrid cultures, clones and subclones Of 19 clones derived from hybrid culture WK x C1 D (WK-D-1),14showed EBNA-positive cells. Of 40 subclones obtained from three EBNA-positive WK-D-1 clones, 34 contained Attempts to associate presence of a specific EBNA-positive cells. All of the 21 clones dehuman chromosome with the rived from hybrid culture NN x IT-22 (NN-Texpression of EBNA 3) contained EBNA-positive cells. Six of 22 Karyotypic analysis of hybrids with mouse clones derived from hybrid culture KM-2 x IT-22 (KM-2-T-10)were analyzed for presence IT-22 and THO-2 cells showed, in addition to of EBNA-positive cells, and only one clone an entire complement of mouse chromosomes, showed presence of such cells. Of 28 clones de- five to eight human chromosomes, of which rived from hybrid culture G X IT-22 (G-T-7), chromosomes 7, 16, 17, and 21 in hybrids KM1-T-9and KM-2-T-10 and chromosomes 19 and 1 4 contained EBNA-positive cells.
LW-A
AK-
1 GOT-1
GUS GRS
PGM-3
SOD-2
HM-B
I M -1 GALT 2GM-2
PGM- 1
phosphog luconiuta s e- 1 (chromosome 1) isocitra te dehydroqenase-1 (chromosome 2 ) galactose-1-phosphate uridiltransferase (chromosome 3 ) phosphoglucornutase-2 (chromosome 4 ) hexosaainidase B (chromosome 5 ) superoxide dismutase-2 (chromosome 6) phosphoqlucon~utase-3 (chromosome 6) beta-glucuronidase (chromosome 7) glutathione reductase (chromosome 8 ) adenylate kinase-1 (chromosome 9) glutamate oxaloacetic transaminase-1 (chromosome 10) lactate dehydrogenase A (chromosome 11)
Symbols used for the isozvme ( 2 8 ) :
C6 PD
NP MPI HEX-A APRT GX PEP-A PH I ADA SOD- 1
ESD
LDH -3 ENO- 2 PEP-B
lactate dehydroqenase B (chromosome 12) enolase-2 (chromosome 12) peptidase B (chromosome 12) esterase D (chromosome 13) nucleoside phosphorylase (chromosome 14) mannosephosphate isomerase (chromosome 15) hexosaminidase A (chromosome 15) adenine phosphoribosy-transferase (chromosome 16) galactokinase (chromosome 17) peptidase A (chromosome 18) phosphohexose isomerase (chromosome 19) adenosine deaminase (chromosome 23) superoxide dismutase-1 (chromosome 21) glucose-6-phosphate dehydrogenase (X chromosome!
Expression of human lsozymes in clones and subclones of WK D-1 hybrid
TABLE 2
HUMAN NPC AND EBV I N SOMATIC CELL HYBRIDS
X in hybrid JN were more frequently represented than other human chromosomes. The hybrid NN-"-3 and its clones showed the presence of 8 to 12 human chromosomes including chromosomes 5, 7, 10, 12, 17, 19, 20 and 21. Hybrid culture G-T-7 (derived from NPC propagated in nude mice) and its clones contained 80-100% cells staining positively for EBNA. Karyotypic analysis of metaphases and isozyme analysis of this hybrid indicated t h e presence of 12 human chromosomes: 1, 5, 6, 9, 14, 15, 16, 17, 18, 19, 20, and 21. A detailed analysis of WK-D-1 hybrid culture clones and subclones for the presence of EBNA, and t h e expression of human forms of isozymes, is shown in table 2. Results of karyological analysis confirm the isozyme results. Of five WK-D-1clones, two were EBNA-negative; results of isozyme analysis of these clones, C3/I and C8/I, are shown in table 2. Isozyme analysis of t h e three EBNA-positive clones, D12II1, G-4/11, and G8/I (table 2) indicated the presence of the following human chromosomes: 12 (ENO-2, PEP-B, and LDHB), 17 (GK), 20 (ADA), and 21 (SOD-1). Of eight subclones derived from clone G8/I, five contained EBNA-positive cells and three did not (fig. 1).Human chromosomes 12 and 21 were present in cells of three EBNA-negative and three EBNA-positive subclones. In addition, two EBNA-negative subclones contained human chromosome 20. All subclones contained human chromosome 17 and one of these subclones (G8/1-3), which contained 8090% EBNA-positive cells and only human chromosomes 17 and 21, was then subcloned. Three of the 14 resulting subclones contained EBNA-positive cells, but there was no correlation between the retention of human chromosomes 17 and 21 and expression of EBNA. In addition, one EBNA-positive subclone, G8/I-6
Human chromosomes 12
EBNA
21
t
-
4-
-
+
3
2
3
2
-
3
0
3
0
Fig. 1 Lack of association between the presence of
EBNA and expression of either human ENO-2 (chromosome 12) or SOD-1 (chromosome 21) in eight subclones derived from clone G8/I (see details in table 3).
5
(table 21, showed the presence of human chromosomes 12 and 17, but not 21. Finally, another EBNA-positive subclone, G8/I-10, showed the presence of only human chromosome 17 (table 2). Similar patterns were observed in one subclone, G4/II-12, derived from clone G4/II and two subclones, D12/11-1and D12/II-14,derived from clone D12III (table 2). All subclones contained EBNA-positive cells and retained, in addition to a n entire mouse complement, any combination of human chromosomes 12, 17, 20 and 21. Back selection in Brd U
To exclude the role of human chromosome 17 in t h e maintenance of EBNA, cells of clones G8/I-10 and D12III were transferred to medium containing BrdU (Croce, '77; Croce et al., '731, and the surviving colonies were propagated for three passages in BrdU medium before analysis. All four subclones established after back selection of clone GEYI-10 in BrdU became EBNA-negative and lost expression of human galactokinase. On the contrary, as shown in table 2, none of the nine BU subclones of clone D12/II expressed the human form of galactokinase, which indicates loss of human chromosome 17 and all these showed presence of EBNA-positivecells in proportions varying from 40-100%. All D12/II-derived subclones expressed human ENO-2 and three clones expressed the human form of PEP-B, which indicates the presence of the proximal fragment of the long arm of human chromosome 12 (McKusick and Ruddle, '77). In addition, four subclones expressed the human form of ADA which indicates the presence of human chromosome 20, and one subclone expressed SOD-1, which indicates presence of human chromosome 21. Clone D12III-BU-6, which contained 40.50% of EBNA-positive cells and expressed only human ENO-2 and PEP-B (table 21, was subcloned. As shown in the ideogram (fig. 3) 10 of the 13 subclones contained EBNA-positive cells and three did not. All 13 cultures expressed human ENO-2, indicating that in all cultures part of human chromosome 12 was present. EBVgenome equivalents in NPCand hybrid cells The results shown in table 3 indicate that, in general, the number of EBV-DNA equivalents per cell decreased in hybrid cells as
6
STEPLEWSKI, KOPROWSKI, ANDERSSON-ANVRET AND KLEIN TABLE 3
Equivalents of EBVgenomes per cell in human parental NPCcells and their hybrids Cells Parental NPC
"(935595)'
Hybrid mass cultures
Clones
EBV genome equivalents
Subclones
WK (159866)' WK-D-1
4
G4/II G8/I
11 10
G8II-3 G8II-4 GWI-9 GIII-I1 G8/I-14
KM-2 (163859)' AM (166628)' EJ (94057)'
G (nude)'
12 - 2 < 1 5 < 1 23 21 26 104 2 30 < 1 40 < 1 25 3 25 5 4 7
KM-1-T-9' KM-2-T-10' AM-T-11 ' EJ-H-1 '
JN (170788)'
+ + + + + + + +-
22 5 5
NN-T-3 '
KM-1 (165836)'
EBNA
JN-H-1 ' G-T-7 '
++
+
+
+
+
+
' See table 1 and text. *Seetable 2 and text. After more than one year in culture, these hybrids have recently become EBNA negative. EBV-DNA equivalents were assayed after hybrids became EBNA negative.
compared to their corresponding parental (patient's) tumor cells. WK-D-1 and G-T-7 hybrids are the exceptions since there was anincrease in t h e amount of EBV genome equivalents per cell in comparison to the parental NPC cells. All hybrid cells that contained EBV-DNA were found t o express EBNA, and the EBNA-negative subclones of hybrid WK-D-1 contained less than one EBV genome equivalent per cell. In two cases (KM2-T-10 and AM-T-111, hybrids that were EBNA-positive at the beginning have recently become EBNA-negative and contain less than one EBV genome equivalent per cell. Regardless of t h e presence or absence of EBV-DNA, all hybrids contained human chromosome(s). DISCUSSION
Although human chromosomes 12 and 21 (or parts of them) seemed to be preferentially retained in EBNA-positive hybrid cells, the results of subcloning these hybrids indicated that neither chromosome 12 nor 21 segregates with EBNA (fig. 1). Analysis of subclones of
Human chromosomes
17
21
EBNA
+
-
f
-
+
3
0
3
0
-
10
1
11
0
Fig. 2 Lack of correlation between presence of EBNA and expression of human SOD-1 (chromosome 21) and GK (chromosome 17) in 14 subclones derived from G8/I-3, an EBNA-positive subclone of WK-D-1 hybrid cultures between NPC and C1 1D cells (see text).
hybrid subclone G8/I-3 (fig. 2), which showed the presence of human chromosomes 21 and 17 as the only human chromosomes, confirmed the fact t h a t neither chromosome 21 nor 17 segregates with the expression of EBNA. Furthermore, in spite of the results obtained with back selection of clone G8/I-10, the possibility that human chromosome 17 maintains EBNA
HUMAN NPC AND EBV IN SOMATIC CELL HYBRIDS
7
Human chromosome 12
EBNA
-k
10
0
-
3
0
Fig. 3 Lack of correlation between presence of EBNA and expression of human ENO-2 in 13 subclones derived from t h e EBNA-positive DlB/II-BU-B subelone of the WK-D-1 hybrid culture between NPC and C1 1D (see text).
has been eliminated by examination of the subclones of hybrid DlBIII, which were backselected in BrdU and which expressed EBNA in the absence of human chromosome 17. Since all these subclones expressed human ENO-2, i t was hoped t h a t analysis of subclones of BU-6 (fig. 3) would permit the discovery of a more positive association between chromosome 12 and the presence of EBNA; the results proved the contrary and, as shown in figure 3, this association was not established for 3 out of 13 clones examined. Whether a fragment of human chromosome 12 was present in the three EBNA-negative subclones of clone G8/I (fig. 1) but could not be detected by isozyme analysis is impossible to exclude. Thus, in the light of these results, it is impossible to correlate the presence of EBVDNA and the expression of EBNA with a specific human chromosome(s), and we have to examine alternate mechanisms of maintaining EBNA in NPC-mouse hybrid cells. We may postulate that production of EBNA is associated with the presence of a fragment of a human chromosome carrying EBV viral DNA sequences which cannot be detected either by karyotype analysis or isozyme assays. Within t h e context of presently available information, it is not possible to disprove this rather unlikely hypothesis. The second possibility is that EBV is present in NPC cells in the form of free plasmids and, hence, can be found in NPC mouse hybrids regardless of the presence of a given human chromosome. However, data obtained by Falk e t al. (manuscript in preparation) indicate that one hybrid clone, NN-T2-7 may contain only integrated EBV-DNA sequences. A third possibility is that the expression of EBNA is due to the integration of t h e viral DNA sequences in the mouse chromosome. I t is clear from data presented in table 3 t h a t there is a positive correlation between
the presence of EBV-DNA and the expression of EBNA in the hybrid cells. On the other hand, there are hybrids that contain human chromosome(s) but are negative for EBNA and do not contain EBV-DNA. In contrast to the results obtained in studies of SV40-transformed human cells (Khoury and Croce, '75; Croce et al., '75) assignment of the gene(s) responsible for the maintenance of the malignant phenotype may present a much more difficult task in NPC hybrids. There is no reason, however, why these studies should not be pursued, since the study of interspecies somatic cell hybrids with a variety of human tumors could ultimately result in the identification of a human chromosome responsible for malignancy. ACKNOWLEDGMENTS
The NPC biopsies were collected and kindly supplied to G. K. by Doctor Surjit Singh (Department of Head and Neck Surgery, Kenyatta National Hospital, Nairobi, Kenya). The expert technical assistance of Maria Obrocka, Hanna Pragert and Marie Prewett is gratefully acknowledged. This investigation was supported, in part, by USPHS Research Grants CA-10815 from the National Cancer Institute and RR-05540 from the Division of Research Resources and by Grant 5 R 0 1 CA-14054-05 from t h e National Cancer Institute and a grant from the Swedish Cancer Society. LITERATURE CITED Croce, C. M. 1977 Assignment of the simian virus 40 integration site to chromosome 17 in t h e SV40-transformed human cell line GM54VA. Proc. Natl. Acad. Sci. (U.S.A.1, 74: 315-318. Croce, C. M., D. Aden and H. Koprowski 1975 Somatic cell hybrids between mouse peritoneal macrophages and simian virus 40 transformed human cells. 11. Presence of human chromosome 7 carrvine simian virus 40 eenome in cells of tumors induced b i h y i r i d cells. Proc. Natl. Acad. Sci. (USA.), 72: 1397-1400. Croce, C. M., A. J. Girardi and H. Koprowski 1973 Assign-
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STEPLEWSKI, KOPROWSKI, AN1DERSSON-ANVRET AND KLEIN
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